Method for producing an implantable ligament and tendon repair device

ABSTRACT

Compositions and blends of biopolymers and bio-acceptable polymers are described, along with the use of benign solvent systems to prepare biocompatible scaffolds and surgically implantable devices for use in supporting and facilitating the repair of soft tissue injuries.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of PCT InternationalApplication No. PCT/US2018/057412, filed Oct. 24, 2018; which is relatedto U.S. Provisional Patent Application Nos. 62/707,159, filed Oct. 24,2017, 62/714,367, filed Aug. 3, 2018, and 62/718,694, filed Aug. 14,2018, the content of which is hereby incorporated by reference in itsentirety.

STATEMENT OF US GOVERNMENT SUPPORT

This invention was made with government support under DARPA ContractHR0011-15-9-0006. The US government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to biopolymer scaffolds and implants for themanagement, protection and repair of injuries to soft tissue injuriessuch ligaments and tendons. The implants have improved physicochemicaland biological properties including enhanced biocompatibility. Theinvention also relates to processes for the production of such implantsusing benign solvent systems.

BACKGROUND

Surgical repairs number around 800,000 annually in the US alone forligaments and tendons of the foot and ankle (for example, Achillestendon), shoulder (for example, rotator cuff), and knee (for example,anterior cruciate ligament), yet the current standards of care involvingthe implantation of replacement and supporting elements are generallyconsidered by medical practitioners to be less than optimal.

Leading ligament and tendon repair graft products intended to providebiocompatible soft tissue support scaffolds often involve two decadesold technologies that in some instance rely on cadaveric tissue orinvasive autografting. Allografts are supply-limited, promote scarformation, may provoke an immune response, and have poorly definedturnover rates, all of which inhibit healing. Autografting also extendssurgery time and associated trauma, and often adds a second costlyprocedure to recover the autologous tissue.

For example, the GRAFTJACKET® Regenerative Tissue Matrix is a sheet-likeproduct formed from donated allograft human dermis, asepticallyprocessed to remove cells and then freeze-dried,http://www.wright.com/footandankleproducts/graftjacket. ArthroFLEX®Decellularized Dermal Allograft is a similar acellular dermalextracellular matrix, https://www.arthrex.com/orthobiologics/arthroflex.

Various other approaches have been taken to develop synthetic orsemi-synthetic components for implantable devices useful as scaffolds tofacilitate repair of, or to support or replace damaged soft tissues suchas tendons and ligaments. Such products must function in a variety ofchallenging biomechanical environments in which multiple functionalparameters must be addressed, among them, for example, arecompatibility, strength, flexibility and biodegradability.

Among such approaches and products are those disclosed, for example, byRatcliffe et al., U.S. Pat. No. 9,597,430 (2017), entitled “SyntheticStructure for Soft Tissue Repair”. This patent describes varioussynthetic fibrillar structures, such as a woven mesh and single ormultilayer planar fibrillar forms. According to Ratcliffe, thesestructures can be made from any biocompatible polymer material capableof providing suitable mechanical properties, bioabsorbable or not.Collagen and lactide are mentioned as suitable. Synthasome's “X-Repair”medical device appears to be related to this patent and has been grantedFDA 510(k) clearance by the US Food and Drug Administration (FDA),(http://www.synthasome.com/xRepair.php).

Another approach is described by Qiao et al., “Compositional and inVitro Evaluation of Nonwoven Type I Collagen/Poly-dl-lactic AcidScaffolds for Bone Regeneration,” Journal of Functional Biomaterials2015, 6, 667-686; doi:10.3390/jfb6030667. This article describeselectrospun blends of Poly-d,l-lactic acid (PDLLA) with type I collagen.Various blends are described with ratios of 40/60, 60/40 and 80/20polymer blend by weight (PDLLA/Collagen). Qiao described a co-solventsystem, and reported that chemical cross linking was essential to ensurelong term stability of this material in cell culture. According to Qiao,scaffolds of PDLLA/collagen at a 60:40 weight ratio provided thegreatest stability over a five-week culture period.

The use of constructs for muscle implants is also described by Lee etal., U.S. Pat. No. 9,421,305 (2016), “Aligned Scaffolding System forSkeletal Muscle Regeneration.” The patent discusses an anisotropicmuscle implant made of electrospun fibers oriented along a longitudinalaxis and cross linked to form a scaffold. Cells are seeded on the fibersto form myotubes. The fibers may be formed from natural polymers and/orsynthetic polymers. Natural polymers include, for example, collagen,elastin, proteoglycans and hyaluronan. Synthetic polymers include, forexample, polycaprolactone (PCL), poly(D-L-lactide-co-glycolide) (PLGA),polylactide (PLA), and poly(lactide-co-captrolactone) (PLCL). The fibersalso may include hydrogels, microparticles, liposomes or vesicles. Whenblended, the ratio of natural polymer to synthetic polymer are between2:1 and 1:2 by weight.

Electrospun scaffolds for generation of soft tissue replacements aredescribed by Sensini et al., “Biofabrication of Bundles of Poly(lacticacid)-collagen Blends Mimicking the Fascicles of the Human AchillesTendon,” Biofabrication 9 (2017) 015025,doi.org/10.1088/1758-5090/aa6204. Two different blends of PLLA andcollagen were compared with bundles of pure collagen.

Yang et al., US Published Patent Application 2014/0011416 (2014), “ThreeDimensionally and Randomly Oriented Fibrous Structures,” describes amethod for producing randomly and evenly oriented three dimensionalfibrous structures via electrospinning. It describes electrospinning adope comprising one or more polymers, such as collagen, polylactic acid(PLA) and others, a solvent and a surfactant. The surfactant can be oneor more of a diverse group including anionic surfactants, cationicsurfactants, nonionic surfactants and zwitterionic surfactants. Thespinning dope also includes one or more of a variety of solventsincluding acetic acid, chloroform, dimethyl sulfoxide (DMSO), ethanol,methanol and phosphate buffered saline.

And Dong et al., U.S. Pat. No. 8,318,903 (2012), “Benign Solvents forForming Protein Structures,” describes methods for forming variousprotein structures by dissolving a protein, such as collagen, in abenign solvent comprising water, alcohol and salt. It also describesconventional electrospinning techniques.

Elamparithi et al., Indian published patent application IN640CHE2013(2013), “A Method for Preparing a Three-Dimensional Collagen Fiber MatUsing Benign Solvent and Products Thereof,” describes a threedimensional, electrospun collagen mat prepared with a combination ofacetic acid and DMSO as an environmentally benign solvent system.Another article by Elamparithi and colleagues uses the solvent system ina process of forming electrospun gelatin. See, for example, “GelatinElectrospun Nanofibrous Matrices for Cardiac Tissue EngineeringApplications,” International Journal of Polymeric Materials andPolymeric Biomaterials 66(1):20-27 (2017).

SUMMARY OF THE INVENTION

The invention relates to a method for producing an implantablebiopolymer scaffolds for use to contribute to, encourage, facilitate andsupport the repair of a soft tissue injury both biologically andmechanically, such as damage and injuries to tendons and ligaments.

Contemplated tendons that may be repaired include the Achilles tendon,rotator cuff tendon, patellar tendon, biceps tendon, and quadricepstendon. Contemplated ligaments than may be repaired include the anteriortalofibular ligament, medial collateral ligament, posterior cruciate andthe ligaments of the spine and temporomandibular joint.

Biopolymer scaffolds according to the invention are produced bydissolving a biopolymer, and optionally a bio-acceptable polymer, in aDMSO solvent system. Preferred solvent systems are mixtures of about 40to 100% by volume of dimethylsulfoxide (DMSO) and about 0 to 60% byvolume of a solvent selected from the group consisting of ethanol,tetrahydrofuran and acetic acid. After preparing a solution of thebiopolymer in the solvent system, biopolymers are generated andcollected.

In certain embodiments of the method and certain embodiments of thebiopolymer scaffold, the biopolymer fibers are formed of collagen,either entirely, or in a range of about 10 to 100% by weight. The use of100% collagen in the biopolymers is preferred. Correspondingly, when100% collagen is not utilized, about 0 to 90% by weight of abio-acceptable polymer is also used to generate the fibers. Contemplatedbio-acceptable polymers are PDLA, PDLLA, PLGA and mixtures thereof.Contemplated types of collagen include type I collagen, atelocollagen,telocollagen, recombinant human collagen and mixtures thereof. And highmolecular weight PDLLA also is preferred, for example, PDLLA having aninherent viscosity of about 1.6-2.4 dl/g.

In various embodiments of the invention, the biopolymer fibers aregenerated by various techniques that including electrospinning andpneumatospinning.

Other aspects of the invention include the use of such implantablebiopolymer scaffolds to encourage, facilitate and support the repair ofa soft tissue injury. Such scaffolds may be formed of one sheet of thebiopolymer fibers mentioned above or from a plurality of sheets. Thefibers range in composition as described above.

Preferred embodiments of the invention reflect these chemicalcomponents, techniques for generating biopolymer fibers, andpost-processing steps for the scaffolds that include vacuum drying toremove residual solvents and annealing of the sheets and scaffolds whilerestraining them from shrinking or while mechanically drawing them alongthe axis of their alignment.

Accordingly, embodiments of scaffolds processed as disclosed, possess anaverage porosity of about 50 to 150 microns as determined by mercuryporosimetry, and in other embodiments about 80 to 120 microns or 100microns. These embodiments also may have an absorbance of about thescaffold's weight in blood in about 5 min and an absorbance of abouttwice its weight in blood in about 20 minutes when measured in vitro; anaverage fiber diameter in the range of about 150-4,500 nm, preferablyabout 300-3,000 nm, more preferably about 500-2,000 nm and mostpreferably about 700-1,200 nm; substantial in vivo cell infiltrationinto the scaffold within about two weeks following implantation into asubject, and in some embodiments the amount of cellular infiltrationreaching to about the full thickness of the implanted scaffold; andwhere the average configuration of the pores and void spaces in thescaffolds is substantially in the shape of a slit relative to otherconfigurations such as elliptical, cylindrical or random poreconfigurations.

Other embodiments of the implantable biopolymer scaffolds have an innersurface formed of substantially aligned biopolymer fibers and an outersurface having fibers that are not substantially aligned or are orientedrandomly.

Yet other embodiments of the invention are implantable biopolymerscaffolds that are seeded with various types of cells. Contemplatedcells include tenocytes, myoblasts, myocytes, satellite cells,fibroblasts, osteoblasts, chondrocytes, and vascular cells, such asendothelial cells and stem cells.

Further embodiments of the invention include the production ofbiopolymer implants in useful dimensions and configurations and packagedin a sterile container.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D show a preferred embodiment of the invention in which abiopolymer scaffold in the form of a sheet is implanted and wrappedaround a partially torn tendon after a surgical suture repair and thenthe scaffold itself is sutured in place. FIG. 1A shows a partially torntendon. FIG. 18 shows a suture repair. FIG. 1C shows an implant wrappedaround repair. FIG. 1D shows an implant sutured in place.

FIG. 2 shows a general illustration of the electrospinning techniquedescribed herein.

FIGS. 3A-3D depict a preferred embodiment of the pneumatospinning deviceof the claimed invention.

FIGS. 4A and 4B show, respectively, a biopolymer scaffold annealed in analuminum frame, and multiple scaffolds processed simultaneously.

FIG. 5 shows scaffold thickness and density pre- and post-annealing.

FIG. 6A shows an H&E histology of subcutaneous implants ofcollagen/biopolymer electrospun from hexafluoroisopropanol (HFP) at 8weeks with low cell infiltration; FIG. 6B shows an H&E histology ofsubcutaneous implants of collagen/biopolymer electrospun from DMSO at 2weeks; and FIG. 6C depicts FIGS. 6A and 6B graphically. * and dashedlines in FIGS. 6A and 6B indicate and demarcate the electrospunimplants, 10× magnification.

FIG. 7 shows the results of 7 day media stability testing of electrospuncollagen:PDLLA scaffolds. FIGS. 7A and 7B depict the electrospunscaffold shrinkage in media and electrospun scaffold volume swell inmedia, respectively. DMSO:EtOH composition: 150 mg/mL 30:70TeloCollagen:PDLLA dissolved in 65:35 DMSO:EtOH. DMSO:THF composition:150 mg/mL 30:70 TeloCollagen:PDLLA dissolved in 75:25 DMSO:THF. HFPcomposition: 100 mg/mL 30:70 TeloCollagen:PDLLA dissolved in HFP.Scaffold area and volume were evaluated at days 0 (prior to incubationin DMEM at 37° C. with 5% CO₂), 1, 4, and 7.

FIGS. 8A-8D show scanning electron microscopy (SEM) of alignedpneumatospun collagen at 30x× (FIG. 8A), 500× (FIG. 8B), 3,000× (FIG.8C) and 10,000× (FIG. 8D) magnification.

FIGS. 9A-9C show a comparison of aligned collagen fibers. Scanningelectron microscopy revealed the potential for generating isotropic andanisotropic collagen fibers by pneumatospinning (FIG. 9A, FIG. 9B), ascompared to aligned electrospun collagen generated using electrospinning(FIG. 9C). Although a wider range of fiber sizes were produced viapneumatospinning, both pneumatospinning and electrospinning producedfibers with 200 nm average diameter (FIG. 9D). Fiber alignmentquantified using ImageJ showed a greater degree of alignment inelectrospun compared to pneumatospun fibers (FIG. 9E).

FIG. 10 shows material properties of pneumatospun and electrospuncollagen. Genipin crosslinked collagen manufactured by electrospinningand pneumatospinning methods were mechanically tested and compared(n=6). Pneumatospun collagen surprisingly had a significantly higheraverage tensile strength and peak load compared to the electrospun group(* indicates p<0.05), with no statistical difference in the modulus orstrain at break found between the two tested groups.

FIG. 11 shows ASC metabolic activity over time (AlamarBlue). ASCs seededon genipin-crosslinked electrospun and pneumatospun scaffolds at 5×10⁴cells/cm² were incubated for two weeks in cell culture media at 37° C.Increasing metabolic activity over time shows cytocompatibility of bothmatrices.

FIG. 12 shows shrinkage of biopolymer sheets that are not annealed orrestrained.

FIG. 13 shows scanning electron microscopy (SEM) images of annealed andas-spun (gripped and loose) scaffolds after 14 days in culture.

FIG. 14 shows fiber alignment at days 0 and 14 for biopolymer scaffoldswith or without annealing or restraining.

FIG. 15 shows fiber diameters at days 0 and 14 for biopolymer scaffoldswith or without annealing or restraining.

FIG. 16 shows a comparison of subcutaneous implant of scaffoldselectrospun out of various solvents to analyze cell infiltration atvarious time points.

FIG. 17 shows rat subcutaneous implants with respect to cellularinfiltration over 2 to 16 weeks, comparing implants with high and lowporosities.

FIGS. 18A-18C illustrates tenocyte cellular alignment and cellelongation along the collagen-based microfibers.

DETAILED DESCRIPTION

The invention relates to implantable biopolymer scaffolds and methodsfor their use in the management, protection and repair of soft tissueinjuries. The injuries preferably involve ligaments and tendons that donot exhibit a substantial loss of tissue as a result of the injury. Theimplants encourage and facilitate a healing response in the area ofinjury. This includes the remodeling of the implant through cellularinfiltration and tissue ingrowth, the deposition of collagen fibers, aswell as vascularization and resorption of the implant by the treatedsubject. FIG. 1 shows a preferred embodiment of the invention in which abiopolymer scaffold in the form of a sheet is implanted and wrappedaround a partially torn tendon after a surgical suture repair and thenthe scaffold itself is sutured in place.

The invention also relates to methods for producing a biopolymerscaffold, meaning a construct formed from biopolymers and bio-acceptablepolymers. Such constructs are preferably substantially aligned fibersformed into layers, mats, sheets and tubes, and may be used as animplant for the management, protection and repair of injuries to softtissue injuries such ligaments and tendons.

A method of the invention dissolves a biopolymer, and optionally abio-acceptable polymer, in a DMSO solvent system comprising about 40 to100% by volume of dimethylsulfoxide (DMSO) and about 0 to 60% by volumeof a solvent selected from the group consisting of ethanol andtetrahydrofuran (THF) to form a biopolymer solution; generatesbiopolymer fibers from the biopolymer solution; and collects thebiopolymer fibers to form a biopolymer scaffold.

Biopolymers

Biopolymers which may be used in a method of the invention to produce abiopolymer scaffold are proteins which are components native tissuesbiological structure and extracellular matrices. Contemplatedbiopolymers are naturally occurring, protein-based macromoleculenatively found in connective and other soft tissue and in theextracellular matrix, such as collagen, elastin, extracellular matrixproteins, fibrin, fibrinogen, gelatin and laminin, and combinationsthereof. Also contemplated are the use of recombinant and chemicallymodified forms of the foregoing protein-based macromolecules, as well ascollagen from marine sources such as jellyfish, sea cucumber andcuttlefish.

A preferred biopolymer is collagen. Type I collagen used forbiocompatible scaffolds according to the present invention generally areextracted from mammalian tissues, particularly bovine and porcinetendons, although recombinant collagen also may be used. Acellular humandermis is sometimes used as a source of collagen.

Type I collagen has been utilized and commercialized in both researchand clinical grade products in two common forms. The more commoncollagen variants, produced with acid and enzymatic digestion of atissue with pepsin, are a form of collagen referred to as“atelocollagen,” as it lacks the end-terminal regions of the collagenprotein (terminal peptide sequence of “DEKSTGISVP vs.pQLSYGYDEKSTGISVP), whereby the telopeptides are cleaved to aid inrecovery of collagen from the parent tissue. Less commonly, collagen issolubilized in mild acid to collect the collagen in solution,maintaining the telopeptides in the monomers of collagen, known as“telocollagen.”

Acid-soluble (telocollagen) and pepsin-soluble (atelocollagen) freezedried collagen are appropriate starting materials for use in a method ofthe invention. A preferred GMP-grade, type I collagen from bovine coriumis available in its native form from Collagen Solutions,http://www.collagensolutions.com/products/medical-grade-collagen.Collagen is also available from other suppliers and from variousspecies, for example, Sigma-Aldrich,http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/structural-proteins/collagen.html.

A preferred scaffold according to the invention is comprised of 100%biopolymer without including a bio-acceptable polymer. Other preferredembodiments produce scaffolds that are mixtures or blends of biopolymertogether with one or more bio-acceptable polymers.

Bio-Acceptable Polymers

A biopolymer scaffold may be produced according to the invention using amixture of a biopolymer and a bio-acceptable polymer. Incorporating abio-acceptable polymer presents a means to modulate overall graftmaterial properties such as strength, fiber size, stability anddegradation characteristics, while reducing overall graft cost forpossibly little loss in cytocompatibility. A wide variety ofbio-acceptable, for example, biodegradable and bioactive, polymers havebeen considered for use in soft tissue repair, alone or in blends withother polymers, and sometimes including components of native tissue suchas the proteins recited above. Useful and improved biomechanical andbiodegradability properties result from the blended combination of suchproteins with various bio-acceptable polymers, for example by combiningcollagen with polylactic acid, including both its L- and D-isoforms, andparticularly so with its racemic mixture referred to as poly-DL-lactideor PDLLA.

The PLLA isoform alone is relatively strong but brittle rather thanelastic. It persists in vivo for about 36 to 48 months. A preferred PLLAfor some applications of supporting injured soft tissue is availablefrom Sigma Aldrich.http://www.sigmaaldrich.com/content/dam/sigma-aldrich/articles/material-matters/pdf/resomer-biodegradeable-polymers.pdf.However, PLLA is insoluble in DMSO, so the use of this bio-acceptablepolymer and a base solvent should be assessed on a case-by-case basisand appropriate solvents other than DMSO generally should be utilized.The PDLA isoform is more elastic and not as brittle, and typically lastsfor 12 to 18 months in vivo. A preferred PDLA is available from SigmaAldrich.http://www.sigmaaldrich.com/catalog/product/SIGMA/67122?lang=en&region=US.PDLLA generally lies between PLLA and PDLA in terms of strength andstability, and in terms of lifespan in vivo is in the range of about 18to 36 months, which generally is long enough to be resorbed and shortenough to avoid encapsulation. PDLLA is an amorphous polymer formed viapolymerization of a racemic mixture of L- and D-lactides. The precisecomposition of the polymer determines its mechanical properties andhydrolysis characteristics.

PDLLA with an inherent viscosity ranging from about 0.5-5 dL/g may beused to produce biopolymer scaffolds according to the invention. A PDLLAhaving a relatively higher average inherent viscosity, about 1.5-6 dL/g,is a preferred bio-acceptable polymer, more preferably about 4-5 dL/g, 5although PDLLA with a lower inherent viscosity, 0.5-1.3 dL/g, can beused when a lower peak stress is appropriate. A preferred PDLLA, havingan inherent viscosity of 1.6-2.4 dL/g, is available from Polysciences,http://www.polysciences.com/default/polydl-lactic-acid-iv-20-28dlg. Alower inherent viscosity PDLLA (IV of 1.3-1.7 dL/g) is available fromEvonik,http://healthcare.evonik.com/product/health-care/en/products/biomaterials/resomer/pages/medical-devices.aspx.A PDLLA with even lower average inherent viscosity of 0.55-0.75 dL/g isavailable from Sigma-Aldrich,http://www.sigmaaldrich.com/cataloaproduct/sigma/p1691?lang=en&region=US;and PDLLA from other sources also is available. And a preferred PDLLAwith a GM P level of purity is available from Corbion (“PURASORB PDL45”) with a relatively high inherent viscosity of 4.5 dL/g.http://www.corbion.com/stabadownloads/datasheets/31d/PURASORB %20PDL%2045.pdf.

Bio-acceptable polymers that may also be useful for a given product ordevice, or when combined with, for example, collagen, includepolylactide, polycaprolactone (PCL) and poly(lactic-co-glycolic acid)(PLGA). Other useful bio-acceptable polymers are known to personsskilled in the art, for example, poly(glycolic acid), polyesters,trimethylene carbonate, polydioxanone, caprolactone, alkylene oxides,ortho esters, hyaluronic acids, alginates, synthetic polymers fromnatural fats and oils, and combinations thereof.

Bio-acceptable polymers used with the biopolymers may be pretreated withone or more functionalization reagents to prepare the bio-acceptablepolymer for cross-linking upon generation of biopolymer/bio-acceptablepolymer fibers from the biopolymer solution. For example, PDLLA can befunctionalized through aminolysis to add amino groups. See, for example,Min et al., “Functionalized Poly(D,L-lactide) for Pulmonary EpithelialCell Culture,” Advanced Engineering Materials 12(4):B101-B112 (2010) athttp://onlinelibrary.wiley.com/doi/10.1002/adem.200980031/abstract.Alternatively, PDLLA can be functionalized by plasma treatment tointroduce carboxylic and amino groups in the matrix.

As a general approach, by way of example, PDLLA can be functionalizedwith OH groups prior to electrospinning. PDLLA pellets are soaked in asolution mixture of 10 mM-1M sodium hydroxide dissolved in 10-20%ethanol in milliQ (ultrapurified) water. The pellets will soak for 10-60minutes at either room temperature or 37° C. Following incubation, thepellets will be rinsed in milliliters of distilled or more highlypurified water and air dried in a biosafety hood. The functionalizedPDLLA chips could then be dissolved in a DMSO solvent system, such asDMSO/ethanol, described below.

When a mixture of biopolymers and bio-acceptable polymers are used, themixture may contain about 10 to 50% biopolymer by weight, preferablyabout 15 to 40% biopolymer, more preferably about 20 to 35% biopolymer,more preferably about 27.5 to 32.5% biopolymer and most preferably about30% biopolymer with the remainder being bio-acceptable polymer. Mixtureof two or more biopolymers and/or two or more bio-acceptable polymersmay be used as the biopolymer and/or as the bio-acceptable polymercomponents.

A preferred biopolymer to bio-acceptable polymer mixture contains about10 to 50% collagen and about 50 to 90% bio-acceptable polymer by weight,preferably about 25 to 35% collagen, more preferably about 27.5 to 32.5%collagen and most preferably about 30% collagen and 70% bio-acceptablepolymer by weight. Type I collagen is preferred and a lactide polymer,particularly PDLLA, is preferred as the bio-acceptable polymer.Telocollagen and atelocollagen are also preferred in such mixtures. Apreferred composition is about 30% type I bovine dermal collagen andabout 70% PDLLA that is not chemically crosslinked during postproduction processing. Such compositions exhibit desired biomechanicalperformance and biostability parameters.

Benign Solvent Systems

A method of the invention dissolves a biopolymer, and optionally abio-acceptable polymer, in a solvent system, preferablydimethylsulfoxide (DMSO), to form a biopolymer solution. A preferredDMSO solvent system contains about 100% by volume of DMSO. Otherembodiments contain about 40 to 100% by volume of dimethylsulfoxide(DMSO) and about 0 to 60% by volume of a solvent such as the monohydricalcohols, cyclic ethers, branched chain ethers and their chlorinated andfluorinated derivatives and esters and combinations thereof.Contemplated solvents include methanol, ethanol, propan-2-ol,butan-1-ol, pentan-1-ol, hexadecane-1-ol and other saturated straightand branched chain hydrocarbons containing a single hydroxyl functionalgroup, and combinations thereof. Preferred cyclic ethers include oxalate(otherwise known as “tetrahydrofuran” or “THE”), oxetane, andcombinations thereof. Preferred solvents are ethanol and tetrahydrofuran(THF), and mixtures thereof, when electrospinning is the technique usedto produce the biopolymer scaffolds. DMSO and acetic acid, and mixturesthereof, are preferred when pneumatospinning is the technique used toproduce the biopolymer scaffolds.

The DMSO solvent systems used in the methods of the invention are“benign” solvent systems. They are solvents capable of dissolving thebiopolymers and bio-acceptable polymers making up a biopolymer scaffold,and are either generally recognized as safe by the US Food and DrugAdministration or otherwise causes minimal risk to the health of a humanor other mammalian subject relative to other conventional solvents usedin electrospinning techniques to produce related implantable scaffolds,for example, such as 1,1,1,3,3,3 hexafluoro-2-propanol (HFP).

DMSO is a polar chemical that readily dissolves various biologicalmolecules such as proteins and nucleic acids. In general, DMSO has beenshown to be a versatile chemical that enhances biological function suchas membrane penetration, membrane transport, anti-inflammation,vasodilation and much more. Moreover, DMSO is a solvent that can be usedin a solvent system or solvent blend with ethanol and THF. It exhibits arelatively low toxicity and is considered to be a safer option ascompared, for example, to HFP, which is a common solvent used forelectrospinning collagen and other polymers. Because of HFP'scomparative toxicity, its presence in an electrospun material or implantadversely impacts biocompatibility after implantation. As is known topersons skilled in the art, DMSO is hygroscopic, so minimizing exposureto water is important. DMSO solutions for preferred solvent systems alsohave a relatively low water content.

DMSO solvent systems for producing biopolymer scaffolds according to theinvention preferably contain about 100% DMSO. Other preferredembodiments which contain DMSO and a solvent contain about 40 to 100%,more preferably about 50 to 99%, 55 to 95% or about 60 to 90% and mostpreferably about 70 to 85%, 75 to 85% or about 80% DMSO by volume andabout 0 to 60%, more preferably about 1 to 50%, 5 to 45% or about 10 to40%, and most preferably about 15 to 30%, 15 to 25% or about 20% byvolume of a solvent selected from ethanol and tetrahydrofuran (THF).Ethanol is hygroscopic and typically kept sealed to minimize moisturecontent. THF is flammable and highly volatile. Exposure to air (that is,oxygen) should be avoided to reduce the possibility of peroxide buildup.Absolute ethanol is the preferred form of ethanol. When using THF, mostpreferred is 25% and when using ethanol, most preferred is 35%.

DMSO solvent systems used in a method of the invention may be preparedby simple mixing or other means known in the art. For example, combiningan appropriate amount of DMSO and either THF or ethanol in a 20 mLscintillation glass vial, and mixing them by gently swirling or bypipetting the mixed solution up and down until solvents are blended. Themixed solutions should be stored in airtight vials and inside a fumehood or other similar device. Long term storage is not recommended, aspossible evaporation will result in changes in solution concentrations.Persons skilled in the art will be able to utilize these and otherbenign solvent systems.

Biopolymer Solutions

In a method of the invention, a biopolymer solution is prepared bydissolving a biopolymer or a biopolymer/bio-acceptable polymer mixturein a DMSO solvent system, for example 30% (300 g/mL) collagen and 70%(700 g/mL) PDLLA by weight (w/w). The biopolymer orbiopolymer/bio-acceptable polymer mixture may be dissolved in the DMSOsolvent system using means known in the art. For example, a solution ofDMSO and ethanol or DMSO and THF is pre-mixed to make the DMSO solventsystem prior to adding the biopolymer and bio-acceptable polymer, ifpresent. Next, the biopolymer such as collagen is added at the same timeas any bio-acceptable polymer, which can be either vortexed together atroom temperature or left sitting at room temperature and inverted byhand prior to generating biopolymer fibers, for example byelectrospinning. Generally, bio-acceptable polymers require vortexing oragitation to dissolve whereas collagen alone does not. Alternatively, abiopolymer such as collagen and bio-acceptable polymers can be dissolvedseparately and brought together or blended together and dissolved at onetime.

Production of Biopolymer Fibers and Biopolymer Scaffolds

The biopolymer fibers after being generated from solution are thencollected to form a biopolymer scaffold. The biopolymer fibers andbiopolymer scaffold may be composed of a single biopolymer, a mixture ofbiopolymers, a mixture of a biopolymer and a bio-acceptable polymer, ora mixture of two or more biopolymers and one or more bio-acceptablepolymers. Biopolymers and bio-acceptable polymers, together with theirpreferred embodiments, are discussed above. The biopolymer fibers andscaffolds each represent separate embodiments of the invention andexhibit reduced or comparable amounts of shrinkage relative to scaffoldsprepared with conventional solvent techniques as well as improvedswelling characteristics when wetted with various liquids afterproduction, for example blood and other biological fluids to which thescaffolds are exposed after implantation in a subject for medicalpurposes.

Biopolymer fibers and scaffolds of the invention may be produced byvarious techniques. Electrospinning and pneumatospinning are preferred.Electrospinning offers relatively more control of both collagenmicrofiber diameter and for controlling the ordering of the fibers intwo dimensions. Electrospinning, however, has limited ability to producethick three-dimensional materials due to electrical insulation of thecollector, typically producing biopolymer sheets of about one millimeteror less in a single layer. This limitation is not present withpneumatospinning which has a theoretically unlimited thickness. We mayalso e-spin with salts such as sodium acetate in the biopolymer mix andthe pH of DMSO can be lowered through a variety of methods such asadding HCl or acetic acid to the DMSO, in order to adjust or improve thestrength or crosslinking of resultant scaffolds, as may be determined bya person skilled in the art.

Electrospinning

Electrospinning is a preferred processing technique to generatebiopolymer fibers from the biopolymer solution. FIG. 2 shows a generalillustration of the electrospinning apparatus described herein. Otherapproaches to separating the blend from the solvent system will be knownto persons skilled in the art, for example, pneumatospinning, extrusion,cold drawing or casting.

Electrospinning is a fiber production technology that draws chargedthreads of polymer solutions or polymer melts into fibers of variousdiameters and lengths. Electrospinning shares characteristics of bothelectrospraying, conventional solution dry spinning and extrusion, orpulltrusion of fibers. Electrospinning of collagen has been widelydescribed as a one-step process for the formation of fibrous materialsthat mimic native tissue structure. Biopolymer scaffolds in the form ofsheets may be produced by electrospinning biopolymer fibers onto ahigh-speed drum (at a surface speed of around 1-20 m/s). As mentionedbelow, the biopolymer scaffolds can be vacuum dried afterelectrospinning to remove residual solvents. For example, the scaffoldscan be dried at a temperature of about 30-37° C. to remove residualprocessing solvents. Electrospinning equipment is conventional andreadily available. Generally, fibrous sheets are readily peeled orremoved from the drum as large sheets which can then be cold-drawn orcut or folded to produce scaffolds of various dimensions.

Pneumatospinning

Pneumatospinning is another preferred fiber and scaffold productiontechnology useful to produce biopolymers and implants according to theinvention. This embodiment of the invention provides an original methodof collagen microfiber production and assembly using high velocity air(pneumatospinning) to generate anisotropic and isotropic scaffolds, oruseful for collagen coatings on other devices. As illustrated in theaccompanying FIGS. 3A to 3D, scaffolds are produced by ejectingbiopolymer solution through an injector 702 (airbrush) into the internalpost fiber collector 706 as the internal post fiber collector 706rotates with respect to the position of the nozzle 704 of the injector702.

A. Internal Post Fiber Collector

In one example implementation of the invention, the collector 706 issubstantially an open cylinder in shape with a hollow interior portion709 bounded on its circumference by a collector wall 716. In otherexample implementations of the invention, the collector can have othercurvilinear planes and/or can be closed at an end opposite the injector.

As shown in FIGS. 3A-3D, fiber collector 706 can be substantially anopen cylinder in shape with internal spokes 710 positioned axially inthe interior portion 709 of the collector 706. The spokes 710 can becoaxial in the same X-Y plane within the collector 706 or can bearranged in a spiral array staggered throughout the length of thecollector 706 as shown in FIGS. 3A and 3C, where spoke holders 708 arepositioned at different points along the length (Z-direction) of thecollector 706. The spokes 710 can be removably attached to the collector706. In one example implementation of the invention, the spokes 710 canbe attached/secured to the collector 706 on the outside of collectorwall 716 using spoke holders 708. The spokes 710 can be removed from thecollector 706 at the end of the collection process to access thebiopolymer scaffold(s). In other example implementations, the spokes 710can be secured to the collector wall 716 on the interior portion 709 ofthe collector 706 and can also be removed to facilitate access to thebiopolymer scaffold(s) at the end of the collection process. The spokes710 extend across the interior diameter of the collector 706. The spokes710 can have a number of different cross-sectional shapes, includingrectangular, circular, and oval, and can have a bladed cross section aswell. The relative size (e.g., diameter, cross-sectional area, etc.) ofthe spokes can be selected based upon the biopolymer used, the size andshape of the scaffold or grafts to be collected, and otherconsiderations.

B. Injector

As shown in FIG. 3A, the injector 702 includes a biopolymer port 703that receives a biopolymer, such as a collagen in acetic acid, forexample. Injector 702 drives the biopolymer with regulated compressedgas, which is introduced into the injector 702 via compressed gas input705. The injector 702 drives the biopolymer through nozzle 704 into arotating fiber collector 706 used to collect the biopolymer scaffold(s).

C. Rotation Components

In the FIGS. 3A-3D, the apparatus 700 uses rotation components 720,including drive motor 714, drive wheel 712, first roller axle 734, firstroller wheels 724, second roller axle 736, and second roller wheels 726,to rotate the fiber collector 706. As the injector 702 ejects biopolymersolution into the collector 706, the injector 702 can be moved relativeto the cross-section A-A of the collector 706 as shown in FIGS. 3A and3B. For example, the injector 702 can be moved in the X-direction withrespect to the circular cross section of the collector, in theY-direction with respect to the circular cross section of the collector706, and in the Z-direction with respect to the circular cross sectionof the collector 706. The injector 702 may be moved in any combinationof the X, Y, and Z directions as well.

Rotation components 720 cause the collector 706 to rotate about its(longitudinal) central axis (Z-direction). Drive motor 714 rotates andengages drive wheel 712, which can be positioned coaxially along driveshaft 713. Drive motor 714 can be a continuous speed motor or can be avariable speed motor as needed in the particular implementation of theinvention. As drive motor 714 and drive wheel 712 rotate (clock-wise,CW, for example in FIG. 3D), drive wheel 712 engages first roller wheels724 mounted on a first roller axle 734, which in turn rotates collector706, which in turn rotates second roller wheels 726.

First roller axle 734 and second roller axle 736 extend longitudinallyand are substantially parallel to the length (Z-direction) of thecollector 706. First roller axle 734 and second roller axle 736 havefirst roller wheels 724 and second roller wheels 726, respectively,mounted coaxially along the central axis of the respective axles 734,736. First roller axle 734 and second roller axle 736 are spaced apartfrom each other (in the X-direction) such that first roller wheels 724and second roller wheels 726 support collector 706 as shown in thefigures. A plurality of first roller wheels 724 and second roller wheels726 can be used. As shown in FIG. 3C, first roller wheels 724 and secondroller wheels 726 are positioned along their respective axles 734, 736such that they do not hit or impair the rotation of spoke holders 708,which can extend through the wall 716 of the collector 706. Spokeholders 708 hold the spokes 710 in position on (and within) thecollector 706.

D. Component Positioning

As shown in the FIGS. 3A-3D, the injector 702 is positioned at one endof the collector 706. As the injector 702 drives the biopolymer throughnozzle 704, drive motor 714 rotates drive wheel 712, first roller axleand wheels (734 and 724), and ultimately collector 706. The position ofthe injector 702 (and nozzle 704) in the X, Y, and Z-directions relativeto the collector 706 and to the spokes 710 in the collector 706 can becontrolled and varied by a plotter position controller (not shownseparately) that moves the injector 702 in the X, Y, and Z-directionsrelative to the collector 706 and to the spokes 710 in the collector706. Similarly, the angle (reference numeral 0) with respect to thecollector inlet 707 at which the biopolymer is ejected from the nozzle704 of the injector 702 can also be controlled and varied by the plotterposition controller. The plotter position controller determines theposition and angle at which the biopolymer solution is emitted/ejectedthrough the nozzle 704 of the injector 702 into the collector 706.

The rotation speed of the collector 706 can be controlled and variedbased on the relative diameters of the drive wheel 712, roller axles734, 736, roller wheels 724, 726, and collector 706 as well as by therotational speed of the drive motor 714 itself, using a speed controller(not shown separately). In this fashion, the pneumatospinning biopolymerscaffold manufacturing apparatus 700 of the invention can position thespokes 710 of the collector 706 at an optimal point (optimal points) inspace to receive and collect the biopolymer fibers from the injector702.

Using the pneumatospinning biopolymer scaffold manufacturing apparatus700 in accordance with the invention, anisotropic or isotropic fibrousgrafts can be collected with higher output, lower cost, and lesscomplexity relative to electro spinning. Collagen microfiber synthesisin this fashion has many applications in medical device manufacturing,including ligament, tendon, and nerve repair as well as for applyingmicrofibrous collagen-based coatings and other biopolymers to othermaterials.

In a pneumatospinning embodiment, the biopolymer solution may be thebiopolymer and/or bio-acceptable polymer dissolved in a DMSO solventsystem or dissolved in acetic acid. The defect-free production rate ispreferably about 2 g/hr relative to 0.0625 g/hr with electrospinning ofcollagen as dissolved in acetic acid and has the potential to increaseto at least 8 g/hr using this approach along with higher efficiencyfiber collecting device engineering. Pneumatospinning is thus a markedlyscalable approach to biopolymer fiber generation while requiring lessspecialized and less expensive equipment.

Processing of Fibers and Scaffolds

A method of the invention utilizes various optional steps forpost-processing of a biopolymer scaffold. One post-processing step is todry the biopolymer scaffold to remove residual solvent at least tolevels consistent with requirements of the Food and Drug Administration.The drying may be done by, for example, air drying, vacuum drying,drying in a desiccator, lyophilization, drying under inert gas and likeapproaches. Preferably, the level of DMSO will be reduced to less thanabout 1.5% by weight of the scaffold.

The post-processing of a biopolymer scaffold also may involve chemical,mechanical, physical or thermal post-processing. For example, abiopolymer scaffold produced by a method of the invention can bephysically post-processed such as by thermal annealing with or withoutmechanical drawing, or by a mixture of annealing, drawing, andrelaxation cycles. Such physical post-processing steps can be applied totemper or otherwise alter the material properties of the resultingbiopolymer scaffold, such as by changing fiber diameter, fiberalignment, and void fraction or porosity of the resulting biopolymerscaffold. For example, as shown in FIGS. 4A and 4B, a biopolymerscaffold is annealed in a frame, for example made of aluminum, andoptionally maintained under tension, to promote fiber alignment andimproved mechanical stability. Scaffolds have been annealed in 3different frame types with and without vacuum for up to 24 hours,specifically at 45, 55 and 65° C. The amount of tension should besufficient to avoid or minimize scaffold shrinkage. Such annealing alsosurprisingly and advantageously increases the thickness of a scaffold,as much as by three fold or four fold, which facilitates the clinicaluse of single layer scaffold implants.

In one embodiment, the electrospun scaffolds are prepared by dissolvinga blend of telocollagen (30 mg/ml) and PDL45 (70 mg/ml) for a totalconcentration of 100 mg/ml in 100% DMSO. The scaffolds are placed undervacuum for up to 24 hours (minimum 2 to a maximum of 24 hours).

Such scaffolds then may be annealed in fixed or un-fixed frames attemperatures of about 45, 55, and 65° C. for about 18 to 24 hours. Themechanical stability of the resulting scaffolds is significantly betterwhen annealed at 65° C. for 18 hours. Persons skilled in the art willevaluate changes in scaffold length, which decreases, and in scaffoldthickness, which increases, over time. Preferably, an annealing timefrom about 1 to 48 hours will be optimal, more preferably about 2 to 24hours.

FIG. 5 shows scaffold thickness and density pre- and post-annealing. 20mL of solution electrospun onto a collector drum with 360 cm² surfacearea will result in scaffolds with the following thicknesses: Thenon-annealed scaffolds have an average thickness of 0.3±0.1 mm. Annealedscaffolds have an average thickness of 5.6±1.2 mm. Depending on theannealing setup, the initial scaffold thickness (non-annealed) andpreferences for the use of a given scaffold, the scaffold thickness canbe increased substantially.

A biopolymer scaffold of the invention optionally also may be chemicallypost-processed. For example, fibers within the scaffold that have beenfunctionalized to provide amino groups prior to dissolving in thesolvent system, as described above, may be crosslinked with aldehydes,in general, more specifically with small chain aldehydes, and preferablyglyoxal or with other conventional crosslinking reagents after itsextraction into a scaffold. For example, crosslinkers such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC),N-hydroxysuccinimide (NHS), genipin, glyceraldehyde, gluteraldehyde maybe used. If the bio-acceptable polymer is functionalized with carboxylgroups, then EDC and other carbodiimides may be used for crosslinking.Isocyanates react with both OH groups and amines. Therefore,isocyanate-based crosslinkers may be used to crosslink the OH groups toeach other within, for example, the functionalized PDLLA (linking an OHgroup to another OH group) to improve media stability, strength.Isocyanates also may be used to link collagen to OH groups infunctionalized PDLLA via the NH₂ group (that is, amine group) from thecollagen. Additionally, photo-crosslinkers can be used.

Physical Characteristics of Biopolymer Fibers and Biopolymer Scaffolds

When generated from a DMSO solvent system according to the invention andprocessed as described in this specification, the biopolymer fibers andthe biopolymer scaffolds which they form have novel and unexpectedproperties and characteristics not seen with other biopolymer fibers andscaffolds prepared by other means and methods.

Biopolymer fibers generated from the DMSO solvent system according tothe invention range in diameter from about 150-4,500 nm, preferablyabout 300 nm to 3,000 nm, more preferably about 500 nm to 2,000 nm andmost preferably about 700 nm to 1,200 nm.

Biopolymer scaffolds produced according to the invention haveadvantageous strength profiles based on several contributing factors,including the solvent system, choice of bio-acceptable polymer, and thevarious post-processing steps as discussed in this specification. Forexample, when considering selection of the solvent system, differingratios of DMSO:THF produced significantly differing mechanical strengthprofiles, with a ratio of 75:25 DMSO:THF being significantly strongerthan other ratios. See Table 2 in Example 7 below. Similar results werealso seen with increasing ratios of DMSO:EtOH, such that increasingrelative DMSO concentrations correlated with increasing strengths, witha ratio of about 80:20 DMSO:EtOH being significantly stronger than otherratios. See Table 3 in Example 7 below. Additionally, biopolymerscaffolds spun out of 100% DMSO are stronger than any scaffold spun outof a blended solvent of DMSO:EtOH or DMSO:THF. See Tables 1 and 3 inExample 7 below.

Biopolymer scaffolds produced according to the invention have a peakstress strength about 2.5 to 10 MPa. The biopolymers possess a modulusof elasticity which is substantially like that of human tendons,particularly the Achilles Tendon, which is about 35-750 MPa. Within thatrange, a modulus of elasticity of about 35-200 MPa for the fibers ispreferred. Also, a strain to failure of 50-100% (0.5 to 1.0 mm/mm) astensile strength as tested at 1 mm/s in hydrated condition is preferred.

The implantable scaffolds preferably possess a tensile strength ofgreater than about 5 MPa, a Modulus of greater than about 6 MPa and asuture pull out strength of greater than 0.64 N.

Post-processing by annealing as described advantageously increases theaverage void volume between the fibers within a biopolymer implant fromabout 5 to 10 times that of implants produced by electrospinning withconventional solvents such as HFP. For example, an annealed scaffoldproduced using a DMSO solvent system according to the invention willhave an average pore size, determined by mercury porosimetry, of up toabout 80 to 120 micrometers, more preferably about 90 to 110 micrometersand more preferably about 100 micrometers, compared with an average poresize of about 7 micrometers in a non-annealed scaffold. Persons skilledin the art will understand that this measurement technique determinesporosity by applying controlled pressure to a sample immersed inmercury. The amount of pressure required to force mercury into the poresis inversely proportional to the size of the pores, such that the largerthe pore the smaller the pressure needed to penetrate into the pore. Theporosity of the preferred implants greatly improves cell infiltration invivo, overcoming a longstanding, significant challenge in the field ofelectrospinning products for medical device use.

Additionally, the implants may be seeded with one or more cell types,including autologous, allogeneic or xenogeneic cells. Contemplated celltypes include stem cells or progenitor cells such as mesenchymal stemcells from adipose, bone marrow, or other locations, and placentaderived cells such as cord blood cells and amniotic membrane cells,induced pluripotent stem cells and embryonic stem cells. Musculoskeletalcells such as tenocytes, myoblasts or myocytes or satellite cells,fibroblasts, osteoblasts, chondrocytes, and vascular cells, such asendothelial cells, also may be used. Additionally, other cells types maybe utilized as appropriate to the repair of other tissue types beyondmusculoskeletal tissues, such as dermal, dura mater, adipose, mammaryand other tissue-specific cell types.

Fiber and Scaffold Alignment

In a preferred embodiment of the implant, the orientation of biopolymerfibers will be different on the inner face of the implant adjacent tothe injured tissue and on the outer face of the implant, with a gradientor other transition zone between the inner and outer faces. For example,the biopolymer fibers may be substantially aligned on the inner face ofthe implant, meaning that at least about half of the fibers lying within15 to 20 degrees of a reference in a scaffold are oriented along acommon axis. This implant preferably has a gradient of less alignedfibers through the thickness of the implant toward the outer surface onwhich the fibers are aligned randomly or otherwise are not substantiallyaligned. In other embodiments, one or more inner-facing layers of amulti-layer implant contain substantially aligned fibers and one or moreouter layers of the implant contain fibers that are oriented randomly orare not substantially aligned.

Referring generally to FIG. 1, the foregoing fiber configurationfacilitates suturing while a scaffold or implant is being surgicallyimplanted into a subject and also imparts improved suture retention.This configuration also provides an anti-adhesion barrier on the outersurface and improves the implant's mechanical properties off-axis,meaning off the axis of the injured tissue and off the axis of theorientation of the tissue's own longitudinal fiber components.

Substantial alignment of the fibers in a scaffold may be produced, forexample, by collecting fibers on a drum as described above while it isbeing rotated at a relatively slow speed resulting in the randomdeposition of fibers. The rotation speed can be increased relativelygradually or relatively quickly up to a rapid, or even full rotationspeed, to collect fibers that are deposited in substantial alignment. Inone embodiment, randomly oriented fibers are collected for about sixhours and then the speed of the drum is increased over the next sixhours to its terminal full speed, such as about 15 m/s. Fibers maycontinue to be collected for an additional twelve hours. This kind ofgradient in the orientation of deposited fibers provides a superior andpreferred attachment for sutures. A gradient of about 25 to 33% fromrandom to substantially aligned fibers is preferred.

Functional Characteristics of Biocompatible Scaffolds for Implantation

As described above, the present invention is directed to the use of abenign solvent system to provide synthetic fibers and related sheet-likeand bundled fiber products for tissue engineering, particularly as softtissue supports useful in the repair of damaged tendons and ligaments.For example, according to the present invention, a tissue-engineeredligament and tendon scaffold formed of collagen and a biodegradablepolymer dissolved in DMSO may be used for repair of a damaged Achillestendon.

With respect to scaffolds prepared from the fibers, the scaffold'swettability shows stability in culture media over 7 days of incubationat 37° C. at 100% humidity in 5% CO₂. Generally, seeded cells preferablyshow robust cell attachment, with more than half of the seeded cellsattaching to the scaffold.

Implants according to the present invention also absorb both clottingand non-clotting blood relatively rapidly as compared with implantsproduced, by example, by conventional electrospinning techniques.Absorption may be determined by techniques known in the art asdisclosed, for example, in Rodriguez et al., “Demineralized bone matrixfibers formable as general and custom 3D printed mold-based implants forpromoting bone regeneration” in Biofabrication 8(3):035007. doi:10.1088/1758-5090/8/3/035007 (July 2016). Annealed scaffolds weresubmerged in human blood to assess blood absorption kinetics. Forexample, a scaffold submerged in heparinized blood absorbed 13 times itsweight of ACD blood (blood that clots) and a comparable scaffoldabsorbed 7 times its weight of heparinized blood in about 30 minutes. Apreferred implant will absorb will about one to about four times itsweight in blood in about 5-30 minutes in vitro.

Benign solvent systems and post processing of implants according to thepresent invention permit the production of scaffolds that promotecellular ingrowth that is substantially improved relative to similargrafts that have been electrospun out of HFP, a conventionalelectrospinning solvent, as shown in FIGS. 6A-C. As shown in FIG. 17,enhanced porosity of the scaffolds according to the invention greatlyimprove cellular infiltration of implanted scaffolds. Cell alignment andcell elongation are also substantially improved (FIGS. 18A-18C). Also,the initial retention of growth factors, when they are present on orembedded in an implant, is substantially more like that exhibitednatively by human tendon, particularly, for example, the AchillesTendon, as discussed in more detail below.

Biopolymer Implants for Clinical Use

Sheets of the biopolymer scaffolds optionally may be laminated throughwelding or suturing or sewing. In general, the sheets of biopolymerscaffolds are stacked together in layers. A brief application of heat inthe range of about 30-100° C. may be applied locally to join them.Additional material also optionally may be added into welds to reinforcethe implant material to aid in suture retention. Additionally, anadhesion barrier may be incorporated into the implant. Such barriers maybe composed of a pure polymer backing (facing away, for example, from atendon to which a biopolymer scaffold is to be affixed) in order toprevent extrinsic cell infiltration. The adhesion barrier layer may beelectrospun, cast, foamed, extruded, or produced by other conventionaltechniques.

As preferred embodiments, the invention relates to biopolymer scaffoldsprepared as described above and prepared for implantation into a subject(preferably mammalian and more preferably equine, canine, feline andhuman subjects) in the form of single or multilayer sheet-likescaffolds. In one embodiment, this scaffold is composed of around 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 or more layers of aligned biopolymer scaffold.A single layer scaffold of the invention may be around 4 cm×4 cm, 5 cm×7cm or about 10 cm×10 cm×about 1 mm in thickness. The total thickness ofthe implant will range from about 0.5 mm to about 6.0 mm, preferablyabout 0.6 mm to about 3 mm, more preferably about 0.7 mm to about 1.0 mmand most preferably about 0.8 mm. An alternative embodiment is amultiple layer scaffold of approximately similar dimensions.

As discussed above, the implants may be produced with a randomlyoriented fiber layer on its outward facing surface. Optionally, theimplant also will have a small section of fibers laying in thetransverse plane around the edges to provide additional biaxial supportfor suture retention. Also, optionally, the inner or outer surface ofthe implant can be marked with an arrow or other recognizable shape todistinguish the inner from outer layers.

The implants for surgical use may optionally be packaged dry in a highbarrier, double foil pouch and may be frozen until being thawed beforeimplantation.

EXAMPLES Example 1: Preparing DMSO/Ethanol Solvents and ElectrospinningFibers of Collagen and PDLLA

In 20 mL scintillation glass vials, 45 mg/mL telocollagen and 105 mg/mLPDLLA were dissolved in 6 mL DMSO:EtOH at a ratio of 65:35 for 20 hourson a shaker. (Alternatively, these ingredients may be dissolved inseparate aliquots before being mixed together.) Collagen was obtainedfrom Collagen Solutions (San Jose, Calif.) and PDLLA was obtained fromPolysciences Inc. (Warrington, Pa. Cat#23976). The vials were placed ona VWR Heavy Duty Vortex Mixer (Cat #97043-562) at level 7 until thereagents dissolved, approximately 20 hours later. The solutions werethen electrospun from a 6 mL plastic syringe with plastic luer having adiameter of 11.65 mm; and a 3.9 cm, 25-gauge stainless steel needle ontoa drum (10 cm diameter×15.8 cm width) with six spokes that are 5 cmapart from each other using an electric motor. The drum was elevated 36cm from the floor of the electrospinning box and the solution needle waselevated 19.5 cm above the floor of the box.

The horizontal distance from the needle tip to the plane of the drum was16.02 cm. The sloped distance from the tip of the needle to the midlineof the drum was 23 cm. Directional air flow from a fan is pointed inline with the needle tip and angled in the direction of the drum. 5 mLof solution were spun at a time. The first syringe was spun with a flowrate of 0.8 mL/hr, +15 kV was applied to the needle and −3 kV wasapplied to the drum. The drum speed was 350 rpm. After the first syringewas empty it was discarded, and a 5 mL syringe full of solution wasattached to the luer and spinning resumed. The second 5 mLs were spun ata rate of 0.6 mL/hr, +8 kV was applied to the needle and −8 kV appliedto the drum with drum speed of 350 rpm. A total of 10 mL was spun. Thedrum was left spinning overnight to facilitate drying of the fibers. Thescaffold was removed from the spokes and placed into a desiccator withcalcium sulfate desiccant.

Example 2: Preparing DMSO/THF Solvents and Electrospinning Fibers ofCollagen/PDLLA

In 20 mL scintillation vials, 45 mg/mL telocollagen and 105 mg/mL PDLLAwere dissolved in 5 mL DMSO:THF at a ratio of 75:25 for 16 hours on ashaker. Collagen was obtained from Collagen Solutions (San Jose, Calif.)and PDLLA was obtained from Polysciences Inc. (Warrington, Pa.Cat#23976). The vials were placed on a VWR Heavy Duty Vortex Mixer (Cat#97043-562) at level 7 until the reagents dissolved, approximately 16hours later. The solutions were then electrospun from a 6 mL plasticsyringe with plastic luer having a diameter of 11.65 mm; and a 3.9 cm,25-gauge stainless steel needle onto a drum (10 cm diameter×15.8 cmwidth) rotated with an electric motor and having six spokes that were 5cm apart from each other using an electric motor.

The distances between the needle tip and drum was 16 cm. The flow ratewas 0.9 mL/hr and +18 kV was applied to the needle. The drum wasgrounded and ran at a speed of 1000 rpm. After the first syringe wasempty it was discarded, and a 6 mL syringe with 5 mL of solution wasattached to the luer and spinning resumed. The spin time of 8 hours wasutilized with a temperature of 23.5° C. and a relative humidity of 51%.The drum was left spinning overnight to facilitate drying of the fibers.The scaffold was removed from the drum and placed in the vacuum ovenwith no heat for 3 hours and then placed into the desiccator.

Example 3: Post-Extraction Treatment of Scaffolds

After extraction, the scaffolds were placed under vacuum to aid furtherdrying and removal of residual solvents. They were then stored inside adesiccator. Generally, after extraction, scaffolds are placed undervacuum, for up to 24 hours and then wrapped in foil or placed inside apetri dish before storing.

Example 4: Preparation of Multi-Layer Collagen-Polymer Scaffolds

Two sheets that were each about 0.2 mm thick were laminated by weldingwith a soldering iron at 100° C. or with a short pulse of heat from animpulse sealer. Additional fibers oriented orthogonally were sealed intothe weld to provide reinforcement for suture retention. Persons skilledin the art will understand how to laminate additional sheets, forexample, three, four, five and six sheets may be laminated by welding ina similar manner.

Example 5: Seeding of Human Tenocytes on a Scaffold of ElectrospunFibers

Human tenocytes (5×10⁴ cells/well) are suspended in serum free media andthen seeded on the scaffolds prepared according to Example 3, above.After 15, 30, and 60 minutes in culture, the plates are gently shaken,and the non-attached cells are removed. The number of non-attached cellssuspended in each well are counted, and the percentage of attached cellson each scaffold disk is determined based on the total number of cellsseeded. More favorable cell attachment is found compared with scaffoldsprepared with conventional HFP solvent systems.

Example 6: Shrinkage and Stability Analysis of Biocompatible Sheets

Telocollagen/PDLLA electrospun sheets (dissolved in various ratios ofDMSO:THF, 100:0-75:25 v/v) were tested for stability in culture media at37° C. after about 5 days. The results demonstrated that there was 70%to 80% total area shrinkage within all the scaffolds tested (FIG. 7A).It was also shown that these scaffolds swelled up to 99% to 142% oftheir original size (FIG. 7B). The mechanical strength of thesescaffolds was significantly reduced. See Table 1 below.

Analytical Methods Used in the Following Examples

The following methods were used to test and characterize the collagenfiber scaffolds of Examples 7-14.

Mechanical Testing:

The material properties of hydrated electrospun and pneumatospun (around1.5 cm diameter×4 cm long) scaffolds were tested through uniaxialtensile testing using MTS Criterion, Model 42 (Eden Prairie, Minn.). Allmechanical testing was performed at room temperature. Scaffold diameterand thickness were measured with precision digital calipers and recordedto calculate cross sectional area. Samples were hydrated in Dulbecco'sModification of Eagle's Medium (DMEM) (Fisher Scientific, Hampton, N.H.)for 1 hour and then loaded on the MTS machine with six sample (n=6)pulled for each group.

Scanning Electron Microscopy:

The structure of uncrosslinked pneumatospun and electrospun scaffoldswas analyzed by scanning electron microscopy (SEM). Genipin crosslinkedpneumatospun scaffolds were also assessed after 30 days in DMEM at 37°C., left loose (un-tensioned). Fiber formation, dimensions and matrixalignment was assessed with Orientation J feature of Image) software(NIH, Bethesda, Md.). SEM imaging was performed at Jefferson Labs(Newport News, Va.) using a JEOL JSM-6060 LV microscope (JEOL Ltd.,Tokyo, Japan) with a 20 kV beam intensity.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wasused to compare the collagen feedstock (Collagen Solutions),pneumatospun and electrospun collagen grafts (uncrosslinked, bothdissolved in acetic acid). Gradient gels (3-8%) (Invitrogen, Carlsbad,Calif.) were run at 150 kV on a Xcell SureLock (Invitrogen) gelapparatus. Gels were stained with SimplyBlue™ gel stain (Invitrogen) andthen rinsed with deionized. The gel was then imaged under white light toview the protein bands.

Fourier-Transform Infrared Spectroscopy:

Fourier-transform infrared spectroscopy (FTIR) (Platinum ATR, Bruker,Billerica, Mass.) was used to assess the presence of type I collagen, asdetermined by the three major amide bonds characteristic at 1235, 1560,and 1650 cm⁻¹ wavelengths. Electrospun and pneumatospun scaffolds werecompared to the starting material by assessing peak displacement andsample purity with the Essential FTIR bioinformatics software (Operant,Madison, Wis.).

Circular Dichroism:

A far UV CD (J-815, JASCO) was taken to compare the CD spectra ofpneumatospun scaffolds with electrospun and starting material. CDspectra was obtained using a cuvette path length of 0.1 cm. Samples weredissolved in 50 mM acetic acid at a concentration of 0.5 mg/ml foranalysis.

Statistical Analyses:

One-way analysis of variance (ANOVA) followed by the post-hoc Tukey'smultiple comparison test was used to assess any difference in fiberalignment between groups. Two-way analysis of variance followed by thepost-hoc Tukey's multiple comparison test was used to assess thedifferences in cell viability between groups and over time. An unpairedt-test was used to assess any differences in mechanical strength betweenthe electrospun and pneumatospun collagen scaffolds. A priori, pvalues<0.05 were defined as significant. All tests were performed usingGraphPad Prism 7, and all parameters are expressed as mean±standarderror of the mean (S.E.M.).

Example 7: Inherent Viscosity of the Bio-Acceptable Polymer

Inherent viscosity (IV) of the PDLLA is correlated with overall strengthof an electrospun scaffold when it is prepared using the disclosedbenign solvent system. Choosing a PDLLA with a relatively higher IVleads to an increase in the peak stress and modulus of elasticity of theconstructs while maintaining characteristics of native ligaments, asdiscussed below in Table 1.

TABLE 1 Summary of Tensile Testing Results (as statistical mean ± SD)Telocollagen:PDLLA 30:70 150 mg/mL 100 mg/mL DMSO:EtOH 65:35 DMSO:THF75:25 100% DMSO 100% DMSO Bovine Tail PDLLA IV PDLLA IV PDLLA IV PDLLAIV PDLLA IV PDLLA IV Ligaments 1.3-1.7 1.6-2.4 1.3-1.7 1.6-2.4 1.6-2.44.5 Peak 5.6 ± 2.2 2.7 ± 0.5 4.0 ± 0.3 4.9 ± 0.9 8.3 ± 0.2 8.8 ± 0.613.3 ± 3.7  Stress (MPa) Modulus 6.1 ± 3.1 46.6 ± 22.7 61.4 ± 21.2 93.2± 17.0 186.8 ± 7.0  59.3 ± 8.5  102.9 ± 27.39 of Elasticity (MPa)

Scaffolds spun with differing ratios of DMSO:THF produced significantlydiffering mechanical strength profiles, with a ratio of 75:25 DMSO:THFbeing significantly stronger than other ratios. See Table 2.

TABLE 2 Summary of Tensile Testing Results (as statistical mean ± SD)Telocollagen:PDLLA (IV 1.6-2.4) 30:70 150 mg/mL DMSO:THF 65:35 DMSO:THF75:25 Peak Stress 4.6 ± 0.3 8.3 ± 0.2 (MPa) Modulus of 68.4 ± 10.3 186.8± 7.0  Elasticity (MPa)

Similar results were also seen with increasing ratios of DMSO:EtOH, suchthat increasing relative DMSO concentrations correlated with increasingstrengths, with a ratio of about 75:25 DMSO:EtOH being significantlystronger than other ratios and 100% DMSO resulting in the strongestscaffold. See Table 3.

TABLE 3 Summary of Tensile Testing Results (as statistical mean ± SD)Telocollagen:PDLLA (IV 1.6-2.4) 30:70 150 mg/mL DMSO:EtOH DMSO:EtOHDMSO:EtOH 100% 50:50 65:35 80:20 DMSO Peak  3.3 ± 0.2 4.06 ± 0.32 4.6 ±0.3  8.8 ± 0.6 Stress (MPa) Modulus 56.3 ± 8.9 61.4 ± 21.2 73.6 ± 12.559.3 ± 8.5 of Elasticity (MPa)

Example 8: Electrospinning of Pure Collagen Fibers

Electrospun collagen scaffolds were collected using a high-speed drumwith a surface speed of 10 m/s to generate aligned fibers. Type Iatelocollagen (Collagen Solutions, San Jose, Calif.) was dissolved at250 mg/ml in 40% acetic acid in water for 2-4 hours with gentle rocking.The collagen solution was pumped at 0.2-0.5 ml per hour using a syringepump (NE-4000 Programmable, New Era Pump Systems, Farmingdale, N.Y.)through a 2-inch-long blunt tip 20 G needle. The distance from theneedle tip to the collector (grounded wires spaced 25 mm apart for “airgap” electrospinning) was 10 cm and the needle was charged to +18 kV.

Example 9: Pneumatospinning of Pure Collagen Fibers

An Iwata Gravity feed airbrush (Iwata, Japan) was modified to producepneumatospun collagen fibers (FIG. 3A). An air pressure of 60 psi(pounds per square inch) was used, and the inner needle of the airbrushwas withdrawn approximately 1 mm from the end of the solution emitter toprevent clogging from viscous collagen solution. Up to 500 mg/ml ofclinical grade type I atelocollagen (Collagen Solutions, San Jose,Calif.) was dissolved for 2-4 hours in 20-50 vol. % 40% acetic acid(Sigma-Aldrich, St. Louise, Mo.) in water by shaking. The solutions werethen tested for the ability to form fibers by pneumatospinning.

Collagen fibers were collected on either static grid (2.5 cm squares ofa common 50 ml Eppendorf test tube rack) or were sprayed into a customrotating tube to collect aligned scaffolds. In the custom engineeredrotating tube design, several pairs of perpendicular rods were insertedthrough a tube to catch the passing fibers while the airflow alignedthem in the direction of flow (illustrated in FIG. 3C). The rotatingcollector tube had 2 1/16″ OD parallel stainless steel rods spaced 2.5″apart were placed through the center of a 2″ ID PVC tube. These parallelrods were placed in a rifled pattern down the length of the 30″ tube at30 degree increments. The rods acted to catch fibers as they passedthrough the tube and align them in the direction of flow. The parallelrods were connected to each other by a thin piece of nylon which allowedthem to be removed from the side of the tube. The tube was rotated witha simple DC motor and several idler rollers at 10-20 degrees per second.Rotating the tube allowed for even fiber collection, with the airbrushheld at approximately 45° C. with respect to the tube inlet. Fibers werecollected for 5 minutes of pneumatospinning and used in subsequentexamples after storage in a desiccator containing a Bel-Art Sciencewarereusable cartridge to ensure dryness.

Pneumatospinning from acetic acid solutions with 20-25 wt. % (forexample, as in 450 mg/ml or 500 mg/ml) type I atelocollagen yielded fewfibers. Solutions with 45-50% collagen produced fibers, yet thesolutions were poorly solubilized, very viscous, and resulted indiscontinuous fiber production during pneumatospinning (Table 4). The40% collagen in 40% acetic acid (aq.) was found to optimally produce acontinuous spray of collagen fibers and generate robust sheets ofcollagen with a static collector (FIG. 3B) that was densely coated withfibers by 5 minutes of spraying, thus these parameters were used for allsubsequent experiments in Examples 3-5. The resulting collectedscaffolds weighed 45-50% of the starting material (total milligrams ofcollagen dissolved before pneumatospinning). Pneumatospun scaffolds werecollected at around a 32× increased rate relative to electrospinningfrom acetic acid (Table 5), such as described in U.S. ProvisionalApplication 62/707,159, incorporated herein by reference. Fasterdeposition rates of pneumatospun fibers was achievable at highercompressor pressures but with decreased collection efficiency.

TABLE 4 Collagen Concentration in Acetic Acid Test Matrix forPneumatospinning Collagen Concentration Produced Fibers 150 mg/mL No 200mg/mL No 250 mg/mL Yes 300 mg/mL Yes 400 mg/mL Yes 450 mg/mL Yes 500mg/mL No

TABLE 5 Collagen Fiber Manufacturing Rate Output ElectrospinningPneumatospinning Deposition Rate 0.0625 g/hr 2 g/hr CollectionEfficiency 52% 23%

Collagen fibers (40% acetic acid (aq.), 300 mg/ml atelocollagen)pneumatospun in the rotating tube collection apparatus (FIG. 3C)designed to impart a higher degree of anisotropy to the collected fibersshowed a generally aligned array of fibers under SEM (FIG. 8).Orientation J (Image) plugin for NIH shareware, Bethesda, Md.)quantified the overall significant improvement in fiber alignment inpneumatospinning in the rotating collection tube relative to collectingpneumatospun collagen on a static grid (FIGS. 9A and 9B). However,electrospun collagen microfibrous scaffolds consistently demonstrated asignificantly higher degree of alignment (p<0.05) than that of thepneumatospun scaffolds (FIG. 9C). The average fiber diameter ofpneumatospun relative to electrospun collagen from acetic acid was0.224±0.051 μm, whereas electrospun collagen had an average fiberdiameter of 0.201±0.047 μm (FIG. 9D). Fiber alignment is shown in FIG.9E.

Example 10: Stability and Mechanical Properties of Pneumatospun Collagen

Pneumatospun atelocollagen was not stable in aqueous media withoutcrosslinking, forming a tacky, gel-like film when hydrated, in contrastto complete dissolution of uncrosslinked electrospun fibers placed inaqueous media. To stabilize the electrospun and pneumatospun collagenmatrices (Examples 8 and 9, above) for use in aqueous media, which areotherwise soluble (electrospun) or gel (pneumatospun) when hydrated,genipin was used as a crosslinker. Genipin was chosen for itsestablished low toxicity and existing use in clinically approved medicaldevices.

A protocol by Mekhail et al. was followed to crosslink both electrospunand pneumatospun aligned type I collagen, as described, for example, in“Genipin-cross-linked electrospun collagen fibers,” J Biomater Sci PolymEd. 2011; 22(17):2241-59. doi: 10.1163/092050610X538209. A solution of0.03 M genipin (Sigma-Aldrich) in 97% Ethanol (Sigma-Aldrich) was usedto crosslink pneumatospun and electrospun scaffolds. Pneumatospunsamples were given a short, even twist to improve fiber packing forcrosslinking. During crosslinking, samples were clamped to hold tensionon the fibers and prevent shrinkage and folding upon itself during the7-day and incubation period at 37° C. The genipin-ethanol bath waschecked every day to ensure the graft remained covered in crosslinkingsolution. Genipin crosslinked scaffolds were washed 20 times withPhosphate-Buffered Saline (PBS) (Cellgro, Manassas, Va.) to wash awayresidual, unreacted genipin, producing deep blue scaffolds.

Despite lesser alignment of the fibers relative to electrospun collagenscaffolds (FIG. 9E), pneumatospun collagen from an acetic solvent thencrosslinked with genipin (tested hydrated for 1 hour in DMEM) weresignificantly stronger, at 1.23 MPa±0.11, compared to electrospunscaffolds (FIGS. 10A, C).

There was no significant difference in modulus of elasticity or strainbetween the groups (pneumatospun collagen at 1.45 MPa±0.34) (FIGS. 10B,D). Pneumatospun collagen scaffolds crosslinking with genipin remainedintact for at least 30 days submerged in DMEM at 37° C., left loose(un-restrained), was morphologically altered with an apparentconstriction and coiling of the fibers into spheres on the previouslyfibrous graft.

Example 11: Chemical and Structural Characterization of Electrospun andPneumatospun Collagen

FTIR analyses between the collagen feedstock (unprocessed, freezedried), electrospun and pneumatospun collagen (Examples 1 and 2, above)showed no shifts in the carboxyl and three amide bonds that arecharacteristic of type I collagen, indicating integrity of the primaryand secondary structure. Circular dichroism analyses showedcomparatively little change in pneumatospun collagen relative to thefeedstock collagen, suggesting the pneumatospinning process does notdenature the protein, preserving the native triple helical structure.SDS-PAGE further confirmed the presence of alpha, beta and gamma chainsof collagen present in unprocessed, pneumatospun and electrospuncollagen fibers.

Example 12: Cell Viability of ASCs Grown on Electrospun and PneumatospunCollagen Scaffolds

Tissue culture 96 well-plates were coated with 200 μL of 7%poly(2-hydroxyethyl methacrylate) (PHEMA) (Sigma-Aldrich) to preventcells from attaching to cell culture vessels. Six-millimeter diameterscaffold disks from genipin crosslinked pneumatospun and electrospuncollagen (Examples 7 and 8, above) were cut from the scaffold sheetsusing a tissue biopsy punch. The samples were then disinfected bysoaking in 70% isopropanol for 30 minutes, followed by three ten-minutewashes in PBS. One scaffold disk was used per well. Humanadipose-derived stem cells (ASCs) (ZenBio, Research Triangle Park, N.C.)were mixed and suspended in DMEM. These cells were seeded at a densityof 5×10⁴ cells/well on both the electrospun and pneumatospun scaffolds.Cell viability on collagen-coated wells of a 96-well plate was used as apositive control. Wells were assessed after days 1, 4, and 7 using thealamarBlue™ (BioRad, Hercules, Calif.) viability assay.

After 7 days of culture, the cell-seeded electrospun and pneumatospunscaffolds were fixed and stained to assess cell morphology andattachment. One set of the samples (n=2) was fixed in 4%paraformaldehyde (Thermo Fischer Scientific, Hampton, N.H.) and stainedfor nuclei and actin filaments using DAPI (Vector Laboratories,Burlingame, Calif.) and Alexa Fluor® 594 phalloidin (Thermo FischerScientific), respectively. These stained samples were imaged usingconfocal microscopy (ZEISS Axio Observer Z1 Inverted MotorizedMicroscope, Oberkochen, Germany).

A second set of samples (n=2) was fixed in 2% glutaraldehyde (ElectronMicroscopy Sciences, Hatfield, Pa.) and stained with osmium tetroxide(Electron Microscopy Sciences, Hatfield, Pa.) to image cell morphologyon the collagen scaffolds.

The alamarBlue™ assay performed to assess the metabolic activity of ASCsgrown on pneumatospun scaffolds using acetic acid and electrospunscaffolds using DMSO, with metabolic activity quantified as increasingover time (FIG. 11), indicated cellular proliferation and viability over14 days in culture. Confocal imaging of ASCs grown for two weeks onpneumatospun and electrospun collagen crosslinked with genipin revealthat cells were present throughout the matrices. A confluent layer ofcells was found atop both groups by SEM, collectively indicating strongcell attachment and cytocompatibility for pneumatospun collagen.

The pneumatospun biopolymers are shown to be stable in cultureconditions for a month, yet with an apparent alteration of the fibermorphology and overall graft topography to a condensed and coiledappearance. This may be partially related to the constriction ofmaterial over time as the grafts were not held under tension, along withthe crosslinking driving this apparent fiber constriction and coiling.Other crosslinkers, such as glyceraldehyde and glutaraldehyde, did notexhibit this change in morphology, suggesting a genipin-related effecton pneumatospun collagen. Despite morphological changes to the material,cell metabolic activity and related cell viability was high onpneumatospun fibers though at least 2 weeks of culture.

Example 13: Pneumatospinning Collagen and PDLLA Blended Scaffolds fromDMSO

To assess if collagen could be combined with a biopolymer to produce ablended biomaterial via pneumatospinning, collagen was dissolved inDMSO-based solvent system along with PDLLA in a 30:70 ratio asempirically determined for optimal fiber production. Telocollagen(Collagen Solutions, San Jose, Calif.) was dissolved withpoly-d,l-lactide (Polysciences, Warrington, Pa.) in dimethylsulfoxide(DMSO, Sigma-Aldrich, St. Louis, Mo.), or DMSO and absolute ethanol(Sigma-Aldrich) at 150 mg/mL and collected on a static grid as describedin Example 9, above. While pneumatospinning of collagen alone from DMSOwas not achieved at the tested concentration, PDLLA alone andcollagen:PDLLA blends were able to form produce scaffolds bypneumatospinning from DMSO alone and from DMSO:ethanol co-solvent system(Table 6). Collagen:PDLA ratios ranging from about 10:90 to 50:50 may beused to prepare pneumatospun fibers according to the invention. FTIRanalyses of the pneumatospun collagen:PDLLA confirmed presence of bothbiomaterials in the collected scaffold. The ability to blend collagenwith a biopolymer, such as poly-d,l-lactic acid here, presents furtherpotential applications of a pneumatospinning method according to theinvention.

TABLE 6 Solvent Compatibility for PDLLA:Collagen Pneumatospinning fromDMSO Solvent and Volume Telocollagen Telocollagen Ratio Only PDLLA Onlyand PDLLA DMSO (100%) No Yes Yes DMSO:EtOH (80:20) No Yes Yes DMSO:EtOH(65:35) No Yes Yes

Example 14: Pneumatospun Fibers Using Telocollagen Dissolved in pH3Buffer

Pneumatopsun teleocollagen fibers were produced using the followingprocedure:

Preparation of pH 3 buffer:

-   -   i. Dissolve 1.2 grams of sodium acetate (final 292 mM) into 22.5        ml Acetic acid (47%) and 27.5 ml MilliQ Water. Adjust pH to 3        using Acetic Acid.

Prepared 10 ml of 300 mg/ml of Telocollagen (cut into pieces) in the pH3 buffer in a scintillation vial 24 h prior to airbrushing. Leftovernight on a rocker (speed 5; Tilt 10) after briefly vortexing @ 400rpm for 1 h @ RT. After 24 h, the telocollagen solution was viscous butthe telocollagen had completely dissolved.

On the day of pneumatospinning, the following setup was made,

-   -   ii. The airbrush was cleaned using water, acetic acid, water and        then ethanol before use.    -   iii. The airbrush needle was pulled out so 6 cm of it was        exposed at the end of the airbrush.    -   iv. Set the drum to a medium speed and the air compressor at 60        psi    -   v. Airbrush was held 24 inches away from the drum in a box        setup. A cardboard ramp was set up so the liquid would settle on        the ramp and so the fibers could go off the ramp and collect on        the rotating drum.    -   vi. Fibers collected on the drum at first but then started to        avoid the drum and formed in the surrounding leading to        significant loss.    -   vii. The pressure was then lowered to 40 psi and the drum speed        was changed to lower speeds.    -   viii. The fibers began to form on the drum again, however, there        was significant accumulation of long fibers around the drum in        the box.

Around 9 ml of solution was airbrushed in 1.5 h. However, more than 40%(approximate estimate based on accumulation on the drum) of the fiberswere lost as the air flow to dry the fibers within the box was suckingup a significant amount of fibers.

The fibers on the drum were air dried for 24 h followed by 24 h in thechemical fume hood.

On imaging the fibers, they appeared wet and hence they were vacuumdried for 3 h when on the drum.

Example 15: Cell Attachment, Proliferation and Infiltration

Aligned electrospun scaffolds were produced by blending telocollagen andPDL45 at a ratio of 30:70, respectively and dissolving in 100% DMSO at afinal concentration of 100 mg/ml. This polymer blend was electrospunonto a wire wheel collector in a vertical electrospinning setup.

One set of the scaffolds was restrained in aluminum frames and annealedat 65° C. for 18 hours to promote further fiber alignment and improvedmechanical stability. All scaffolds were cut into 10×30 mm strips andelectron beam (E-beam) sterilized prior to cell experimentation.

To assess human tenocyte cell attachment, one set of each as-spun orannealed scaffolds (n=3) were placed in grips and held under statictension to prevent shrinkage and promote further fiber alignment (thesescaffolds are referred to as ‘gripped’ scaffolds). The available cellseeding area of these scaffolds in the grips is 10×10 mm. Second sets ofeach as-spun or annealed scaffolds (n=3) were cut into 10×10 mm piecesto match the available seeding area of the gripped scaffolds. Thesesamples were placed in ultra-low cell binding culture plates withouttension (These scaffolds are referred to as ‘loose’ scaffolds). Humantenocytes were suspended in serum-free media, seeded at a density of1×10⁵ cells/scaffold and remained in culture for 30 and 60 minutes. Cellattachment on collagen-coated wells was used as a positive control. Ateach time point the scaffolds were removed from the wells and washed 4times by dipping in separate media-containing wells to remove thenon-attached cells. The number of non-attached cells suspended in eachwell was counted and the percentage of attached cells on each scaffoldwas determined based on the total number of cells seeded.

To assess human tenocyte cell proliferation one set of each as-spun andannealed gripped scaffolds (n=6) and one set of each as-spun andannealed loose scaffolds (n=3) were seeded with 25×10³ cells/scaffold.Cellular proliferation was assessed after 1, 7 and 14 days in culture.The alamarBlue™ metabolic activity assay is a standard method to testcompatibility of device with an intended cell type. Healthy andmetabolically active cells will metabolize resazurin from alamarBlue.The metabolism of resazurin (blue in color) reduces it to resorufin (redin color), allowing fluorescent monitoring of metabolic activity in thecell media over time. Both metabolite and the byproduct are non-toxic.AlamarBlue fluorescence level is directly proportional to the number ofviable cells as it measures the metabolic activity of live cells.

Cell infiltration was assessed over a 14-day time frame. One set of eachas-spun and annealed gripped scaffolds (n=3) and one set of each as-spunand annealed loose scaffolds (n=3) were seeded with 1×10⁵cells/scaffold. Cellular infiltration was assessed after 1, 7 and 14days in culture. Cells were stained with DAPI nucleic stain and cellularinfiltration was measured by confocal microscopy. Five fields of viewper each scaffold were investigated. At each field of view, multiple 5μm thick z-scan slices were captured and the total depth of cellinfiltration was calculated based on the number of cell containingslices captured. The total depth was averaged between the five fields ofview.

Statistics:

All parameters are expressed as mean±standard error of the mean (S.E.M).Two-way analysis of variance (ANOVA) followed by the post-hoc Tukey'sMultiple Comparison Test was used to assess the differences in cellattachment, proliferation and infiltration. A priori, p values below0.05 were defined as significant.

Annealed and as-spun scaffolds (gripped and loose) were seeded withhuman tenocytes to assess the ability of these scaffolds to support cellattachment. After 30 and 60 minutes in culture, all scaffolds showedgreater than 50% and 90% cell attachment, respectively. There were nosignificant differences in cell attachment between all conditions testedafter 60 minutes (p>0.05). Additionally, the proliferation of humantenocytes was assessed on gripped and loose, annealed and as-spunscaffolds after 1, 7 and 14 days in culture. All scaffolds that weretested supported cell viability and proliferation. It was shown that asignificantly higher number of metabolically active cells were presenton all scaffolds after 7 and 14 days in culture (p<0.05). The smallernumber of cells within the loose scaffolds is partially due to spillageof a small amount of cell suspension off the scaffolds during cellseeding and mainly due to reduced available growth area as a result ofscaffold shrinkage.

Assessment of cellular infiltration determined that none of thescaffolds promoted significant cell infiltration over a 14-day timeperiod. However, after 7 days in culture gripped as-spun scaffoldsshowed significantly deeper cell infiltration compared to loose, as-spunscaffolds (p<0.05). Additionally, after 14 days in culture grippedannealed scaffolds supported significantly deeper cell infiltration thanloose as-spun scaffolds (p<0.05).

Example 16: Physical Properties of Telocollagen-PDL45 Scaffolds inCulture

Annealed and as-spun telocollagen-PDL45 electrospun scaffolds wereproduced and gripped as described above. FIG. 12 shows shrinkage ofbiopolymer sheets that are not annealed or restrained. After 14 days inculture the annealed and as-spun (gripped and loose) scaffolds wereimaged through scanning electron microscopy (SEM) (FIG. 13). Fiberalignment and fiber diameters were measured using ImageJ software(available at imagej.nih.gov/ij/). These measurements were compared withfiber alignment and diameters of loose scaffolds at day 0 after a30-minute soak in phosphate buffered saline (PBS) (FIGS. 14 and 15).Fiber alignment was measured using the ‘Directionality’ function ofImageJ. This function captures the direction of the fibers and plots ahistogram showing the number of occurrences that a fiber has laid withina certain angle. Therefore, a narrower and higher histogram representsmore fibers within a certain direction.

Statistics:

All parameters are expressed as mean±S.E.M. Unpaired nonparametricKolmogorov-Smirnov t test was used to assess the differences infrequency distribution of fiber alignment. One-way analysis of variance(ANOVA) followed by the post-hoc Tukey's Multiple Comparison Test wasused to assess the differences in fiber diameters. A priori, p valuesbelow 0.05 were defined as significant.

It is shown that annealing under static tension results in further fiberalignment and a decrease in fiber diameter as a result of a scaffold'stendency to shrink. These physical changes are demonstrated by thesignificant differences in fiber alignment and diameter between annealedand as-spun scaffolds at day 0 (FIGS. 14 and 15). Telocollagen-PDL45scaffolds shrink in media at 37° C. However, through annealing orgripping these scaffolds under static tension in culture, scaffoldshrinkage and loss of fiber alignment was substantially reduced. Asdepicted in FIG. 12, it is evident that after 14 days in culture thegripped scaffolds retain their initial size whereas the annealed loosescaffolds only retain about 60% of initial size and the loose as-spunscaffolds shrink to less than 25% of their initial size. Additionally,SEM images depicted in FIG. 13 demonstrate that both annealed andas-spun gripped scaffolds remain highly aligned while both annealed andas-spun loose scaffolds lose their fiber alignment in culture. ImageJfiber alignment analysis of the SEM images revealed that annealedscaffolds at day 0 show significantly higher degree of fiber alignmentthan all other scaffolds tested (p<0.05). It is shown that grippedscaffolds retain their fiber alignment while there is loss of fiberalignment within loose scaffolds. The degree of fiber alignment withinas-spun loose scaffolds was significantly reduced compared to all otherscaffolds tested (p<0.0001). It is apparent that this loss of fiberalignment is due to the shrinkage observed within loose as-spunscaffolds.

Analysis of fiber diameters suggest that annealed scaffolds at day 0exhibited significantly smaller fiber diameters than all other scaffoldstested except gripped as-spun day 14 scaffolds (p<0.05). This decreasein fiber diameter is due to the tendency of restrained scaffolds toshrink under heat treatment. Both gripped and loose annealed scaffoldsexperienced fiber swelling shown by the significant increase of theirfiber diameter (p<0.0001). The significant decrease in fiber diameter ofgripped as-spun scaffolds compared to as-spun day 0 suggests thatrestraining as-spun scaffolds in culture may induce the same physicaleffects as annealing. There were no significant differences in the fiberdiameter of the loose as-spun fibers after 14 days in culture (FIG. 15).

Example 17: Cellular Morphology and Elongation on Telocollagen-PDL45Electrospun Scaffolds

Annealed and as-spun scaffolds were prepared as described above. Bothgripped and loose scaffolds were seeded with 1×10⁵ human tenocytes perscaffold and remained in culture for up to 14 days. To visualize cellmorphology and elongation all the scaffolds cultured with humantenocytes were fixed in 4% paraformaldehyde and stained for nuclei andactin filaments using DAPI and Alexa Fluor® 594 phalloidin,respectively. The stained samples were imaged using confocal microscopy.Percentage of cell elongation was determined based on the averagecalculated nuclei aspect ratio by measuring the length and width oftwenty cell nuclei. Additionally, the degree of cell alignment wasdetermined by measuring the angle of twenty elongated cells in thedirection of elongation over 180° and the frequency distribution waspresented at every 2°. Both measurements were performed using confocalimages taken at 40× magnification.

The results demonstrate that human tendon cells had a higher percent ofelongation within the gripped scaffolds as compared to the loosescaffolds. This is due to higher degree of fiber alignment within thegripped scaffolds as they retain their shape whereas the loose scaffoldslose their fiber alignment due to shrinkage and swelling. The directionof actin filaments (red) demonstrates cell elongation on the grippedannealed and gripped as-spun scaffolds along the direction of thefibers. The random morphology of actin filaments in the cells on theloose scaffolds demonstrates the loss of fiber alignment within thesescaffolds. This random morphology is confirmed by the SEM images of thefibers shown in Example 16, FIG. 13. Cellular spreading and percentelongation were assessed by cell aspect ratio measurements. Bothannealed and as-spun gripped scaffolds supported significantly higherpercent cell elongation when compared to their loose counterparts(p<0.0001 and p<0.01). Additionally, the frequency distribution ofcellular directionality revealed a higher degree of cellular alignmentwithin the gripped scaffolds.

Example 18: Assessment of Heat Treated Tellocollagen-PDL45 ElectrospunScaffolds

Application of heat to enhance stability and structure of electrospunfibers:

-   -   1. Aligned electrospun sheets of feedstock were produced by        dissolving telocollagen-PDL45 (30:70) in 100% DMSO at 100 mg/ml.    -   2. Scaffolds were post processed by:        -   a. Annealing at 65° C. for 18 hours        -   b. Annealing at 65° C. under vacuum for 18 hours        -   c. Drawing to 30% strain at 85° C.    -   3. Scaffolds were assessed for:        -   a. Impact of temperature and vacuum on electrospun scaffolds        -   b. Changes in biochemical and mechanical properties as a            result of heat treatment using XRD, Fourier Transform            Infrared Spectroscopy (FTIR), DSC, Sodium Dodecyl Sulfate            Polyacrylamide Gel Electrophoresis (SDS-PAGE), SEM and            uniaxial tensile testing.

Statistics:

All parameters are expressed as mean±S.E.M. Unpaired nonparametricKolmogorov-Smirnov t test was used to assess the differences infrequency distribution of fiber alignment. Ordinary One-way analysis ofvariance (ANOVA) followed by the post-hoc Tukey's Multiple ComparisonTest was used to assess the differences in peak stress, modulus ofelasticity, % strain at break and peak load. A priori, p values<0.05were defined as significant.

The mechanical properties of as-spun and heat-treated scaffolds wereassessed to evaluate impact of temperature and vacuum on fibers.Mechanical testing indicated that all treatments significantly increasedpeak stress of scaffolds when compared to as-spun control group(p<0.05). Among treated groups, scaffolds drawn at 85° C. showedsignificantly higher peak stress than scaffolds annealed at 65° C.(p<0.05). The heat-treated groups exhibited significantly higher modulusof elasticity than the as-spun control group, however, the scaffoldsdrawn at 85° C. exhibited lower modulus of elasticity than both annealedgroups (p<0.01). Although, peak stress and modulus of elasticity wereaffected by heat treatment there were no significant differencesobserved in their peak load. Both annealing conditions improved strainat break significantly (p<0.05). Scaffolds annealed at 65° C. withoutvacuum exhibited the highest % strain at break (p<0.05). These resultsindicate heat treatment impacts the structure of the fibers and promotesincreased peak stress, modulus of elasticity and % strain at break. Thiscould be due to changes in degree of fiber and molecular alignment.

To determine the impact of heat treatment on the restrained or drawnscaffolds, SEM images were taken and fiber alignment was analyzed usingthe ‘Directionality’ function of ImageJ, as described in Example 16. Theresults show that fiber alignment was significantly improved withinscaffolds drawn at 85° C. or annealed at 65° C. (p<0.05). Although thedrawn electrospun scaffolds under 85° C. exhibit a high degree of fiberalignment, the corresponding SEM image shows that some of the fiberswere broken, therefore, this method may not be ideal for tissueregeneration.

Additionally, the impact of post-processing on the crystallinity ofpolymers was assessed. Telocollagen and PDL45 fiber crystallinity wasevaluated using XRD. The results show that all groups have similarintensity and the peaks overlap indicating that heat treatment did nothave an impact on polymer crystallinity. Scaffolds treated at 65° C.under vacuum had a lower peak intensity and a slight shift to the righton the 2θ axis.

The effect of heat on these scaffolds was also analyzed by FTIR. Theseresults confirmed the presence of collagen within the heat-treatedgroups by presence of amide I (^(˜)1650 cm⁻¹), amide II (^(˜)1560 cm⁻¹),amide A (^(˜)3285 cm⁻¹) and amide B (^(˜)2917 cm⁻¹) bonds. The amide III(^(˜)1245 cm⁻¹) bond could not be easily distinguished as PDL45 has thesame FTIR fingerprint as collagen within this area. Because the peak isslightly shifted from the pure collagen, we cannot fully determine ifthis peak is from the collagen or if it is due to the peak from PDL45.If amide III from collagen is present but the peaks have shifted, itindicates that the state of the bond has changed. However, FTIR showspresence of amides I, II, A, and B bonds, which confirms collagenpresence within all heat-treated groups.

Collagen denaturation of annealed samples was analyzed and compared tocollagen starting material and as-spun control groups. The DSC graphshows two peaks for collagen starting material; a broad peak at ^(˜)90°C. and a sharp peak at ^(˜)200° C. The graph shows as-spun control andannealed groups have shifted to lower temperatures when compared tocollagen starting material. Even though collagen thermal denaturationtemperature decreased after electrospinning, DSC data suggests thatannealing increases denaturation temperature as the peak for theannealed sample is ^(˜)58° C. while as-spun control is ^(˜)55° C.

Another method used to examine the impact of electrospinning, heatannealing, and vacuum drying on collagen chains is SDS-PAGE. As-spun andheat-treated scaffolds were dissolved in acetic acid or DMSO and run onSDS-PAGE. The distinct bands visible at 238 and 117 kDa confirm thepresence of alpha and beta chains, respectively, within electrospunfibers. Moreover, lack of smearing below 117 kDa shows collagen has notbroken down due to electrospinning or any post-process treatments.

Example 19: Subcutaneous Implant Cell Quantification

Subcutaneous implant of scaffolds electrospun out of various solventswere compared to analyze cell infiltration at different time points. Theresult indicates that scaffolds electrospun out of DMSO havesignificantly higher number of cells when compared to HFP scaffolds at 2and 8 week time points after implantation (FIG. 16).

Example 20: Determination of Residual DMSO

GC/MS was used to determine residual DMSO in electrospun scaffolds. Thegrafts that were vacuumed for 2, 4, 6 and 24 hours were compared tografts that were not vacuumed. The GC/MS analysis on the samples wereperformed by Mass Spec Lab, CA, USA using an Agilent 7890A GC and datawas analyzed using the ChemStation software.

Dimethylacetamide (DMAC) was used as the extraction solvent to recoverthe DMSO from the scaffolds. Briefly, about 20 mg of the test sampleswere suspended in 3 mL of DMAC solvent (extraction 1). After heatingthese at 60° C. with agitation for 24 h, the supernatants were used forGC/MS analysis. Duplicate extracts were prepared for each of the samples(duplicates labeled 1 and 2; see Table 7) as well as 2 injections wererun for each duplicate (inj. 1 and inj. 2 in Table 7). A secondextraction step (extraction 2) (with the residue of the same sample) wasconducted to confirm complete recovery of residual DMSO. Upon runningboth extracted supernatants (extraction 1 and extraction 2), it wasobserved that the first extraction was able to extract over 95% of theDMSO. A standard curve for DMSO in DMAC was generated using theintegrated area of the most intense ion peak for DMSO as a function ofDMSO concentration. The extent of DMSO in the unknown samples was thendetermined using this standard curve.

Table 7 shows a summary of the results obtained from the GC/MS study onthe scaffolds. Data in Table 7 clearly indicates that the amount of DMSOextracted from our samples are significantly below (>1000-fold less)than the acceptable range of 50 mg/dose/day, according to FDAguidelines.

TABLE 7 Summary of results obtained from GC/MS study to determineresidual concentrations of DMSO in the scaffolds Average % by % by Massof DMSO Amount of weight weight Standard Sample Run Scaffold (gm) PeakArea DMSO (gm) DMSO DMSO Deviation No vacuum inj. 1 0.0189 106898830.00033 1.770 1.74 0.033 inj. 2 0.0189 10655792 0.00033 1.764 inj. 10.0195 10662179 0.00033 1.710 inj. 2 0.0195 10654148 0.00033 1.709Vacuum 2 hrs inj. 1 0.0202 10805416 0.00034 1.675 1.66 0.020 inj. 20.0202 10805559 0.00034 1.675 inj. 1 0.0188 9960636 0.00031 1.647 inj. 20.0188 9895768 0.00031 1.635 Vacuum 4 hrs inj. 1 0.0209 10823608 0.000341.622 1.65 0.033 inj. 2 0.0209 10834614 0.00034 1.624 inj. 1 0.019210322380 0.00032 1.677 inj. 2 0.0192 10354167 0.00032 1.683 Vacuum 6 hrsinj. 1 0.0189 10055074 0.00031 1.656 1.66 0.009 inj. 2 0.0189 100081450.00031 1.647 inj. 1 0.0203 10816384 0.00034 1.669 inj. 2 0.020310756775 0.00034 1.659 Vacuum 24 hrs inj. 1 0.0202 9723421 0.00030 1.4931.48 0.019 inj. 2 0.0202 9738375 0.00030 1.496 inj. 1 0.0208 97945270.00030 1.462 inj. 2 0.0208 9789379 0.00030 1.461

REFERENCES

All documents identified in this specification, including the followingarticles, are incorporated by reference in their entireties.

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The claimed invention is:
 1. A method for producing an implantableligament and tendon repair device comprising the steps of: dissolvingType I collagen and a bio-acceptable polymer selected from the groupconsisting of PDLA, PDLLA, PLGA, and mixtures thereof, in a DMSO solventsystem comprising about 40 to 100% by volume of DMSO and about 0 to 60%by volume of a solvent selected from the group consisting of ethanol,tetrahydrofuran and acetic acid to form a biopolymer solution;generating biopolymer fibers from the biopolymer solution; collectingthe biopolymer fibers to form a biopolymer sheet; and annealing thebiopolymer sheet; and wherein the biopolymer fibers comprise about 10 to35% by weight of Type I collagen and about 65 to 90% by weight of thebio-acceptable polymer; and wherein the biopolymer fibers in thebiopolymer sheet are not chemically cross-linked.
 2. A method of claim1, wherein the biopolymer fibers further comprise: about 20 to 35% byweight of Type I collagen and about 65 to 80% by weight of thebio-acceptable polymer.
 3. A method of claim 2, wherein the Type Icollagen is selected from the group consisting of atelocollagen,telocollagen, recombinant human collagen and mixtures thereof.
 4. Amethod of claim 3, wherein the biopolymer fibers have an averagediameter of 700 nm to 1,500 nm.
 5. A method of claim 1, wherein thebiopolymer fibers are generated by a technique selected from the groupconsisting of electrospinning and pneumatospinning.
 6. A method of claim2, wherein the biopolymer fibers further comprise about 27.5 to 32.5% byweight of Type I collagen and about 67.5 to 72.5% by weight of thebio-acceptable polymer.
 7. A method of claim 1, wherein the biopolymersheet of the ligament and tendon repair device exhibits one or more ofthe characteristics selected from the group consisting of: (i) anaverage porosity of about 80 to 120 microns as determined by mercuryporosimetry; (ii) an absorbance in vitro of about its own weight inblood in about 5 minutes and an absorbance of about twice its own weightin blood in about 20 minutes; (iii) substantial in vivo cellinfiltration into the scaffold within about two weeks followingimplantation; and (iv) a 4 to 8-fold higher number of adhered cellsafter 2 to 8 weeks following subcutaneous implantation as compared to animplanted device comprised of Type I collagen and bio-acceptablepolymers generated using an HFIP electroprocessing solvent.
 8. A methodof claim 1, wherein the DMSO solvent system comprises about 100% DMSO byweight.
 9. A method of claim 2, wherein the DMSO solvent systemcomprises about 100% DMSO by weight.
 10. A method of claim 6, whereinthe DMSO solvent system comprises about 100% DMSO by weight.
 11. Amethod of claim 1, wherein the bio-acceptable polymer is high viscosityPDLLA.
 12. A method of claim 2, wherein the bio-acceptable polymer ishigh viscosity PDLLA.
 13. A method of claim 6, wherein thebio-acceptable polymer is high viscosity PDLLA.
 14. A method of claim 2,wherein the biopolymer fibers are collected to form a biopolymer sheethaving a thickness that ranges from about 0.5 mm to about 6.0 mm.
 15. Amethod of claim 2, wherein the biopolymer fibers are collected to form abiopolymer sheet having fibers laying in the transverse plane capable ofproviding biaxial support for suture retention.
 16. A method of claim 2,wherein the biopolymer fibers are collected to form a biopolymer sheethaving a first side having substantially aligned fibers and a secondside having fibers that are not substantially aligned.
 17. A method ofclaim 16, wherein the biopolymer fibers are collected to form a gradientof alignment from isotropic to anisotropic of the biopolymer fibersthrough the biopolymer sheet of about 25 to 33%.
 18. A method of claim17, wherein the gradient of alignment is in a layer or zone of theligament and tendon repair device.
 19. A method of claim 2, furthercomprising the step of marking the inner or outer surface of theimplantable ligament and tendon repair device so as to distinguish theinner-facing from outer-facing sides of the device as implanted.
 20. Amethod of claim 2, further comprising the step of packaging theimplantable ligament and tendon repair device in a high barrier pouch.21. A method of claim 2, wherein the biopolymer sheet of the ligamentand tendon repair device exhibits one or more of the characteristicsselected from the group consisting of: (i) a range of tensile strengthof about 4 to 16 MPa; (ii) a modulus of elasticity of about 35-200 MPa;and (iii) a peak stress 2.5 to 10 MPa.
 22. A method of claim 1, whereinthe biopolymer sheet of the ligament and tendon repair device exhibitsone or more of the characteristics selected from the group consistingof: (i) a range of tensile strength of about 4 to 16 MPa; (ii) a modulusof elasticity of about 35-200 MPa; and (iii) a peak stress 2.5 to 10MPa.
 23. A method of claim 1, wherein the biopolymer sheet of theligament and tendon repair device exhibits a range of tensile strengthof about 4 to 16 MPa.
 24. A method of claim 1, wherein the biopolymersheet of the ligament and tendon repair device exhibits a modulus ofelasticity of about 35-200 MPa.
 25. A method of claim 1, wherein thebiopolymer sheet of the ligament and tendon repair device exhibits apeak stress 2.5 to 10 MPa.
 26. A method of claim 5, wherein thebiopolymer fibers are generated by electrospinning.
 27. A method ofclaim 1, wherein the bio-acceptable polymer is PDLA.
 28. A method ofclaim 1, wherein the bio-acceptable polymer is PDLLA.
 29. A method ofclaim 1, wherein the bio-acceptable polymer is PLGA.